Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In particular, we need to know how the protective coats of protein outside and inside the viral surface reacts to the changes in acid that come soon after internalization. In addition, Influenza virus assembles on the plasma membrane where viral proteins localize to form a bud encompassing the viral genome, which ultimately pinches off to give rise to newly formed infectious virions. Upon entry, the virus faces the opposite taskfusion with the endosomal membrane and disassembly to deliver the viral genome to the cytoplasm. There are at least four influenza proteinshemagglutinin (HA), neuraminidase (NA), matrix 1 protein (M1), and the M2 ion channelthat are known to directly interact with the cellular membrane and modify membrane curvature in order to both assemble and disassemble membrane-enveloped virions. 1. The virus that causes flu, influenza, is coated with spike proteins called hemagglutinin (HA) that play a large role in determining its immunogenicity and in the vaccine to flu. This HA protein plays a crucial role during infection after viral endocytosis, undergoing a conformational change that drives the membrane fusion of viral and endosomal membranes at the low pH of the endosome. Although membrane fusion is widely thought to proceed through an intermediate called hemifusion, in fact the hemifusion structure had never been determined. In this project, influenza virus-like particles carrying wild-type HA or an HA hemifusion mutant (G1S) and liposome mixtures were studied at low pH by cryo-electron tomography. For the first time in virology, the Volta phase plate was used, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a lipidic junction. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a ruptureinsertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis. 2. In cells, influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify.. At a pH of about six, the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of about five to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. This year we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. 3. Blast-induced traumatic brain injury (bTBI) continues to be a worldwide health problem. bTBI can be complex, resulting from one or more physical phases of the blast phenomenon. Even those experiencing low-level blast explosions, such as those produced by explosives used to breach fortifications, can develop neurocognitive symptoms without evidence of neurotrauma. The cellular mechanisms of this phenomenon are unknown. The primary phase of bTBI, characterized by organ-shockwave interaction, is unique to blast exposure. Understanding the mechanisms and pathology arising from the primary phase of bTBI is limited, in part, because of the limited availability of in vitro models simulating the blast shockwave. Therefore, it is critical to develop experimental methods to study the primary phase of bTBI. To better study the primary phase of bTBI, we developed a pneumatic device that simulates an explosive blast by producing pressure transients similar to those observed in a free field explosion and is compatible with real-time fluorescence microscopy of cultured cells; this device can produce blast-like pressure transients with and without accompanying shear forces. Using Ca2+ ion-selective fluorescent indicators, changes in intracellular free calcium following simulated blast were detected. We previously showed that a) cultured human brain cells are indifferent to transient shockwave pressures known to cause mild bTBI, b) when sufficient shear forces are simultaneously induced with the shockwave pressure, central nervous system (CNS) cells respond with increased intracellular Ca2+ that propagates from cell to cell; and c) cell survival is unaffected 20 hours after shockwave exposure. In this years project, we report the cell type responsible for the waves of increased intracellular free Ca2+ in dissociated human CNS cultures, and that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human astrocytes, compared to rat astrocytes, had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity.